The early olfactory system, one of the best-characterized sensory systems, is well understood for its connectivity and how it represents odors. However, how odor representation is affected by endogenous neuromodulation remains controversial and elusive. In recent years, neuromodulation in olfactory circuits has garnered great attention in regards to the targets of modulatory cells within these circuits. To date, the manner in which olfactory circuits control the activity of modulatory neurons, and thus modulator release, has been entirely unexplored. Thus, no models presently exist demonstrating the natural pattern of modulator release in olfactory structures, rendering it impossible to study neuromodulation is a natural manner. Furthermore many modulatory neurons, including those involved in olfaction, are also known to possess co- neurotransmitters. The role of such co-transmission remains elusive. This proposal will address these gaps by focusing on the Drosophila antennal lobe, which is analogous to the olfactory bulb, but comprising far fewer neurons than its mammalian counterpart. We will use patch clamp physiology and develop novel approaches to map out the wiring diagram between serotonergic neurons and neurons of the first olfactory relay. Importantly, we will emphasize the feedback connections made from olfactory neurons back onto modulatory cells. Such connections shape activity in modulatory cells and may regulate modulator concentration locally in olfactory circuits. Additionally, we are screening RNAi collections to isolate tools to knock down the expression of individual neurotransmitters in serotonergic neurons that possess multiple transmitters. These RNAi tools will first be combined with optical imaging to determine how each transmitter influences olfactory coding in the antennal lobe. Next, we will use these reagents to alter synaptic function in flies performing an olfactory discrimination task to link sensory coding and perception.